Optimization Of The Fermentation Conditions Of Bacillus Subtilis HS-A38

Bacillus subtilis can synthesize a variety of antimicrobial peptides with broad-spectrum bacteriostatic activity through non-ribosomal pathways.Because of their excellent stability,low resistance to drug resistance,the lipopeptide was widely applicated in cosmetics,food,animal husbandry,and environmental control.In this study,we first studied the antibacterial substances produced by Bacillus subtilis HS-A38,which was preserved in our lab.Firstly,we compared the purification method.It has showed that the crude lipopeptide obtained by the acid precipitation method was more suitable for the later analysis of the experiment than the salt precipitation.The results of layer chromatography?TLC?and the matrix-assisted laser desorption ionization time of flight mass spectrometry?MALDL-TOF?found that the crude extract of this antibacterial lipopeptide was mainly composed with the Bacillomycin,iturin and Fengycin.The physical and chemical properties of the crude extract were then analyzed.On the basis of the above,in order to obtain low-cost,high-yield antibacterial lipopeptides,the fermentation process of strain HS-A38 was optimized.The optimal medium composition obtained through single-factor experiments and response surface experiments.Its components were potato starch,soybean peptone,glucose,soybean meal,MnSO₄,MgCl₂,CaCl₂,ZnSO₄ and K₂HPO₄.The orthogonal experiment was used to optimize the fermentation process.After optimization,the fermentation process was as follows:30°C,180 r/min,60 h,and 6%inoculation in the optimized medium?60 mL/250 mL flask?.TLC and MALDI-TOF mass spectrometry were used to analyze the components of antimicrobial lipopeptides before and after medium optimization.The half-quantitative PCR method was used to analyze the expression levels of several key enzyme genes of lipopeptide during fermentation.The results showed that the expression levels of Ipa-14 and BYA in the new culture medium increased significantly in the early fermentation stage.The results showed that,after optimization,the main antimicrobial lipopeptides were Iturin,Bacillomycin D,FengycinA and FengycinB.The analysis of the physicochemical properties of the antimicrobial lipopeptides and the optimization of the culture medium provide an advantageous reference value for modern low-cost and high-efficiency industrial production of lipopeptides,and provide a certain basis for the extensive use of lipopeptides.